Early and accurate assessment of suspected acute myocardial infarction is critically dependent on the sensitive and specific detection and quantitation in blood, serum or plasma of released cardiac muscle intracellular components in order to distinguish a potentially lethal event in need of emergency measures from non-life threatening conditions such as angina and non-cardiac chest pain such as dyspepsia. Early electrocardiographic changes are neither adequately specific nor sensitive, and the medical profession has come to rely on serum biochemical markers of cardiac tissue injury for early diagnosis. Initially, the serum markers creatine kinase (CK) and specifically the cardiac CK-MB isoform were used, and subsequently myoglobin was identified as a more sensitive early indicator of cardiac damage. More recently, the cardiac troponin complex and its subunits have come to be preferred as markers of myocardial damage because of their high cardiac specificity. These tests, along with other markers of cardiac muscle damage, provide a high degree of diagnostic accuracy. If performed in the emergency room, an early and accurate diagnosis of myocardial damage offers great advantage to a suspected heart attack victim.
Troponin is a muscle protein integrally involved in the calcium-dependent regulation of muscle contraction. Troponin exists in both cardiac and skeletal muscle as a non-covalently-bound complex of three subunits, the isoforms troponin C, the calcium-binding subunit, troponin I, the inhibitory subunit, and troponin T, which locates the troponin complex on tropomyosin. Diagnostic tests in which the level of troponin subunits or complexes is measured in bodily fluids frequently utilize purified troponin subunits or complexes as antigens for the preparation of antibodies used in the assay procedure, as well as the purified subunits or complex used as controls and calibrators in performing the assays. Assay calibrators are used to prepare a series of dilutions by which a standard curve across the operating range of an assay is generated; assay controls are used to confirm that an assay is operating properly by ensuring that the assayed value of pre-determined samples fall within an acceptable range around their labeled values. In order for the assay to be calibrated properly. the troponin controls and calibrators must be stable and in a form which is immunodetectable by the antibody.
Upon cardiac muscle injury and necrosis, troponin leaks from heart tissue into circulation, where its sensitive detection can help diagnose a heart attack. The amino acid sequence differences between the cardiac and skeletal muscle isoforms of the troponin subunits are exploited in diagnostic tests which specifically measure the cardiac isoform of the troponin subunits and complexes. Skeletal and cardiac troponin I share great sequence homology at the C-terminus but differ substantially at the N-terminus enabling the development of cardiac troponin I-specific immunodetection methods.
Diagnostic tests for cardiac troponin I are available, for example, the troponin I Stratus(R) test from Dade International, Inc., the Opus(R) test from Behring, and the Access(R) test from Beckman. The Stratus(R) troponin I assay utilizes a monoclonal capture and monoclonal detector antibody. The Access (R) troponin I assay also utilizes a monoclonal capture and monoclonal detector antibody, and its calibrator/control is a complex of native cardiac troponin I and troponin C. The Opus(R) troponin I assay utilizes polyclonal antibodies as both capture and detector.
Because of the differences in the methodology and components among the above-mentioned assays. and the calibrators/controls cannot be used interchangeably among assays. For example, the Stratus(R) assay detects an N-terminal region of troponin I; its calibrators/controls, which employ an N-terminal peptide from cardiac troponin I, are not detectable in the Access(R) and Opus(R) assays, as the antibodies in these latter assays are not directed to the same N-terminal peptide portion of troponin I. On the other hand, the Access(R) assay controls, which are detectable in the Access(R) assay with the highest level of sensitivity and cut-off value of all three assays, measure about four times higher in the Opus(R) assay. In contrast, the Opus(R) assay controls are detected poorly by the Stratus(R) assay. Values provided by the controls of one manufacturer's assay can only be used in interpreting assays run on that assay. This presents a major problem for the adoption of a universal standard and inter-laboratory comparisons of data.
Furthermore, troponin I in vivo is not a stable molecule and undergoes degradation by proteolytic enzymes present in the body. Although it is somewhat protected from proteolysis by complex with troponin C, this complex is non-covalent and thus is subject to dissociation, rendering troponin I susceptible to proteolytic attack. The same susceptibility is evident in calibrators and controls, whether derived from natural or recombinant sources.
To overcome some of the aforementioned difficulties with the stability of troponin I, commonly-owned U.S. Pat. No. 6,077,676, incorporated herein by reference, describes the preparation of a single-chain polypeptide comprising troponin I and troponin C, which is more resistant to proteolysis. WO 97/19955 describes a 153 amino acid fragment of troponin I containing the N-terminus, prepared by cyanogen bromide cleavage of native or recombinant troponin I, followed by purification, for use as a calibrator or control for troponin I assay. The fragments appears to be around the same size as a degradation product of troponin I present in the serum of a patient that had a myocardial infarction. This troponin I fragment proved to be readily detectable by the components of the aforementioned STRATUS® troponin I assay, as this assay utilizes monoclonal antibodies which recognize epitopes contained within the claimed fragment.
It is toward the improvement of assays for troponin I by providing methods and reagents for detecting a stable, circulating form of troponin I that the present invention is directed.